Oligonucleotides and short RNA molecules cannot easily cross cell membranes because of their size and their polyanionic nature resulting from the negative charge of the phosphate groups. Delivery has therefore been one of the major challenges for RNAi technology (Castanotto and Rossi, Nature 2009; 457: 426-433). To trigger RNAi following systemic administration, a formulation containing short RNA molecules not only requires (1) that the payload be protected from enzymatic and non-enzymatic degradation, and (2) that the appropriate serum half-life and biodistribution of the formulation be provided, but also requires (3) that cellular uptake or internalization of the formulation occurs and (4) and that once internalized, delivery to the cytoplasm of the cell is facilitated. Many formulations that excel in criteria (1) and (2) above show deficiency in criteria (3) and (4), i.e., many formulations show excellent biodistribution but no delivery of formulated agents. Since the oligonucleotides are either not internalized or, once internalized, not released to the cytoplasm, there is no knockdown of the target gene. Importantly, criteria (3) and (4) are equally critical for local delivery (e.g., tissue-specific delivery) as well. Moreover, Lu, Langer and Chen (Mol. Pharm. 2009; 6(3):763-71) postulated that while endocytosis is the primary method of internalization for the nanoparticle formulations in the cell, most of the formulation and payload thus taken up by the cells do not reach cytoplasm and are therefore unable to trigger RNAi. That is, once the formulation is inside the cell, the oligonucleotides pass through the endocytic pathway, eventually being delivered to the lysosome, where the oligonucleotide undergoes lysosomal degradation.
The instant invention is directed to the discovery and design of phase changing charge-trapped peptides that are able to trigger structural changes in a formulation comprising the oligonucleotide and the phase changing charge-trapped peptide, where the structural changes increase release of the oligonucleotide (and, optionally, the peptide, if it is/remains conjugated to the oligonucleotide) to the cytoplasm. Though not being bound by theory, after endocytosis and entry into the endocytic pathway (e.g., the endosome and/or multi-vesicular bodies (MVBs)), the decreased pH of the compartments of the endocytic pathway induces protonation of the phase changing charge-trapped peptide, resulting in the release of the associated oligonucleotide, not only from whatever vesicle or micelle within which the oligonucleotide and associated peptide may optionally have been delivered, but also from the endocytic pathway compartment (e.g., endosome), thereby resulting in localization to the cytoplasm, where the oligonucleotide can be active as, e.g., an RNAi agent, as opposed to transiting to and being degraded within the lysosome.